Objectives: Most patients with high risk HPV DNA and/or abnormal cytology do not develop cervical cancer and are often subjected to repetitive testing and biopsies. The invasive phenotype of oncogenic HPV is dependent upon integration, which is difficult to measure directly. Neoplastic transformation is facilitated through DNA methylation, which silences key viral replication sequences (e.g., capsid protein L1), allowing for expression of the viral oncogenes E6 and E7. Similar epigenetic phenomena may also result in aberrant signaling of the death-associated protein kinase (DAPK) tumor suppressor gene product, further promoting cervical carcinogenesis. We studied oncogenic HPV L1 and DAPK methylation as biomarkers for progression.

Objectives. Although HPV-DNA testing allows for identification of high-risk infections, only a small subset progress to high-grade dysplasia or carcinoma. HPV-16 L1 gene methylation is an epigenetic event resulting from viral integration which can be measured via a high thru-put system. Our hypothesis is that HPV-16/18 L1 gene methylation occurs uncommonly and its detection may serve as a biomarker for high risk disease.

Phylogenetic research in virology addresses the origin and evolution of viruses in time and space, searches for molecular changes that could explain altered pathology or epidemics, and establishes databases for a natural foundation of taxonomy. Some examples are: 1 Influenza virus genome rearrangements and its ability to cross hostspecies barriers helped to explain influenza epidemics (Murphy and Webster 1990). 2 Sporadic occurrences of neurovirulence after polio vaccination were caused by rare point mutations of the attenuated virus (Minor 1992). 3 Evidence that the human immunedeficiency virus-1 is closely related to a retroviral precursor in African primates has been central in the search for the origin of the acquired immunodeficiency syndrome (AIDS) epidemic (Sharp and Li 1988).

This study was performed to elucidate the presence of human papillomavirus (HPV) infection of the vulva by colposcopy, histology and the polymerase chain reaction (PCR). Colposcopy defined 5 patterns of vulval epithelial lesions inconspicuous to the naked eye. Of these 75 subclinical vulval lesions, HPV infection was diagnosed by histology in 20.0% of minor epithelial changes with faint acetowhitening, 52.2% of conspicuous acetowhite lesions, 63.0% of acetowhite areas with satellite lesions, 84.6% of villous lesions, and 85.7% of villous lesions with surrounding acetowhitening. The corresponding HPV DNA positivity rates by PCR were 60.0%, 73.9%, 70.4%, 84.6% and 100% respectively. The oncogenic HPV type 16 was detected by PCR in 37.3% of the samples. These results provide firm evidence for the prevalent existence of subclinical HPV lesions of the vulva. Some of these infections may not produce significant morphologic changes detectable by colposcopy or histology. Subclinical vulval lesions are common and may constitute a reservoir for repeated cervical HPV infections, as well as a source of contamination of cervical samples for HPV DNA detection by sensitive molecular techniques.

In 25 partners of women with genital human papillomavirus (HPV) infection or cervical intraepithelial neoplasia, colposcopic examination revealed the existence of subclinical HPV infection of the male lower genital tract in 22 cases. It manifested either as short papillae tipped with acetowhite changes, or flat acetowhite lesions on the foreskin, glans, periurethral region, scrotum, perineum and/or perianal region. Multiple lesions involving several anogenital areas were common. Some of these abnormalities were small and inconspicuous. Of these 22 cases, 17 had histological evidence of HPV infection. Although Southern blot hybridization detected HPV DNA in only one case, polymerase chain reaction (PCR) analysis revealed HPV DNA in 20 cases There were 10 cases of HPV 16. Subclinical HPV disease is best identified by colposcopy and confirmed by PCR. In treating HPV disease, colposcopic recognition of subclinical HPV disease forms an essential part of the management plan.

The close epidemiological relationships between specific genital human papillomavirus (HPV) types and neoplasia of the cervix uteri have been extensively documented worldwide, including Singapore. Cervical cancer incidence rates in Singapore show variations between the major ethnic groups. To ascertain the corresponding HPV infection rates among the various races in Singapore, we analysed the cervical smears of 225 women by filter in situ DNA hybridization, and compared the data with a previous similar study. Fourteen (6.2%) individuals were HPV-positive, with HPV 16 and HPV 31 being the commonest types. No significant difference between HPV positivity rates in Chinese (5.0%) and in Malays (6.7%) was found, even though Chinese have a higher cervical cancer incidence than Malays. Furthermore, the cervical HPV carriage rate among women with normal cytology was 5.9%. In the light of reports of high genital HPV prevalence rates detected by DNA amplification, these data support the notion that HPV infection is commonly latent and requires the cooperation of other factors for cervical carcinogenesis.

The E6/E7 promoter of all genital human papillomaviruses is responsible for expression of the viral transforming genes. Centered 60 bp upstream of the transcription start, it contains a 20-bp segment with partially overlapping binding sites for the viral E2 proteins and for a cellular factor that was identified by footprint experiments. Bandshifts, bandshift competitions, and footprints revealed that protein complexes between nuclear extracts and these sequences have binding properties indistinguishable from those of the Sp1 factor that binds the simian virus 40 early promoter GC motif. Reactions of these complexes with anti-Sp1 antiserum were analyzed by superbandshifts and precipitation with protein A, and the results confirmed the identity of this transcription factor as Sp1. Sp1 binds in simian virus 40 and different human papillomavirus promoters the consensus sequence 5'-NGGNGN-3'. RNase protection analysis of in vitro or in vivo transcriptions with wild-type and mutant test vectors shows that the E6/E7 promoter of human papillomavirus type 16 is functionally dependent on the Sp1 distal promoter element. In all genital papillomaviruses, the Sp1 hexamer is invariably spaced by a single nucleotide from the distal E2 element, suggesting some precise interaction between Sp1 and E2 proteins. Published experimental evidence documents negative regulation of the E6/E7 promoter by E2 proteins through the proximal E2 element, whereas only minor quantitative differences in E6/E7 promoter function after cotransfection with E2 expression vectors were observed in this study. A detailed study of the interactions of Sp1 and E2 proteins with one another and with the corresponding three binding sites may reveal a complex modulation of this promoter.

Oncogenic viruses serve as useful experimental tools and models of cellular transformation and cancer biology. Tumour virology has made enormous contributions towards the molecular elucidation of malignancy, especially in the concept of oncogenes and anti-oncogenes. Here we consider the criteria, mechanisms and co-factors of viral carcinogenesis. Some viruses associated with cancers in man and animals are reviewed, with special focus on the role of human papillomaviruses (HPV) in cervical cancer. Southern hybridization and polymerase chain reaction (PCR) data demonstrating the presence of genital HPV types 11, 16, 18 and 31 in cervical neoplastic lesions from Singapore patients are presented. Sensitivity standardisation and specificity of the Southern and PCR techniques are emphasised. Common themes in viral oncology are highlighted, such as epidemiological associations; viral type, host and tissue specificities; transforming genetic elements; viral integration; and interaction with co-factors.

Nucleic acid hybridization, and more recently, gene amplification by the polymerase chain reaction, have contributed immensely to the development of molecular genetics. These powerful tools have been greeted and employed with great enthusiasm, in view of their wide-ranging applications in biological research and clinical diagnosis. Here we review the basic principles and some applications of these techniques, particularly in infectious diseases and oncogenesis. In order to illustrate their use, data on specific methods as applied to the detection of human papillomaviruses is presented. Gene probe and DNA amplification technology will undoubtedly continue to provide critical molecular pathways in the biomedical research endeavour.

Human papillomaviruses are found in up to 90% of all cervical carcinomas and are considered to play a causal role in the etiology of this malignancy. The genome of human papillomaviruses consists of a single circular DNA molecule with a size of approximately 8000 basepairs. 90% of this genome encodes proteins involved in functions such as neoplastic transformation of the host cell or formation of the viral capsid. The remaining 10% of the genome, which is termed upstream regulatory region (URR), harbours elements to control expression of the viral genes. We have identified in the URR DNA elements that regulate viral gene expression in the presence of glucocorticoid hormones or tumour promoting substances. This was done by DNase I protection experiments and functional analysis of fusion genes. Our data predict that the transforming potential of the virus might be stimulated by certain steroid hormones, polypeptide hormones and tumour promoting chemicals.

The upstream regulatory region of the human papilloma virus-16 (HPV-16) genomic DNA contains a sequence element with a large degree of homology to the partially palindromic sequence GGTACANNNTGTTCT, which is the consensus sequence of the glucocorticoid responsive elements of known genes regulated by this steroid hormone. DNase I and dimethylsulfate protection experiments reveal the binding of this sequence by rat glucocorticoid receptor protein. A 400-bp DNA segment centrally containing this sequence confers strong inducibility by dexamethasone to the promoter p97 of HPV-16 and to the Herpes simplex virus thymidine kinase promoter, as judged by chloramphenicol acetyltransferase activity and RNase protection assays. The same DNA segment, that does not contain the consensus sequences of all papilloma viruses relevant for E2 protein-mediated transcription enhancement, functions in an enhancer-like fashion in addition to its glucocorticoid responsive action. This hormone-independent transcription enhancement is absent in human MCF7 cells, but is strong in human HeLa cells where the combined activity of the constitutive and the steroid hormone-dependent enhancer elements stimulate transcription by a factor of 500. This cell type specificity of the HPV-16 enhancer may be responsible for the tissue tropism of the virus. These observations and the presence of numerous homologies to known enhancers of cellular and viral genes suggest a complex pattern of activation of the human papilloma virus-16 promoters.

We have constructed and functionally tested a cassette-vector-system for the transcription and translation of open reading frames (ORFs) in cells of higher eukaryotes. The vectors are derived from the plasmid pBR322 and can be selected and amplified in Escherichia coli. Alternative eukaryotic promoters can be inserted between the restriction sites SphI and KpnI, translation initiation motifs between KpnI and BglII, linkers for the adjustment of the translation reading frame and the insertion of genes or gene segments between BglII and HindIII, followed by a HindIII-EcoRI segment with splicing and polyadenylation signals derived from SV40. A prototype vector system, pORFEX11, 12 and 13, contains the strong cytomegalovirus immediately early promoter and a 10-bp motif of the SV40 T-antigen translation start. Polylinkers derived from pUC18 permit the insertion of ATG-less ORFs downstream from the ATG of the vector. Either of the three alternative polylinkers adjusts the appropriate translation frame. A similar construct contains the regulatable promoter of the Drosophila heat shock gene 70. We inserted genes or gene segments, that code for the bacterial chloramphenicol acetyltransferase, the bacterial gene conferring resistance against hygromycin, and the ORF E7 of the human papillomavirus type 18 into these vectors. After transfection of mouse L fibroblasts, all proteins and functions were expressed in accordance with the prediction. In transiently transfected L cells, the E7 protein expressed from pORFEX12 constitutes approximately 2.0% of total cell protein. This E7 protein could be localized by immunocytochemistry as a cytoplasmic component.

We have studied the extrachromosomal maintenance and the transcription regulation of two glucocorticoid-inducible genes on bovine papilloma virus (BPV) vectors in c127 mouse fibroblasts. These genetic elements were the rat tryptophan oxygenase (TOase) gene promoter, which is active in vivo only in hepatocytes, and the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR). From both genes, fusions of the 5′-flanking region of the transcription unit to the bacterial gene for chloramphenicol acetyltransferase (CATase) were constructed. These fusion genes were inserted either into pCGBPV9, a BPV vector encoding G418 resistance, into pBPV-BV1, a vector containing “stabilizing” segments of the human β-globin gene, or into a BPV construct, whose bacterial plasmid sequences could be removed before transfection. Five constructs of the two latter groups, selectable in c127 cells only as foci, were normally maintained in the extrachromosomal state. In contrast, three out of five constructs based on pCGBPV9 and selectable for resistance against G418 were maintained in a high molecular weight form, most probably of intrachromosomal concatemeric nature, while the remaining two G418-resistant constructs appeared alternatively in this or the extrachromosomal monomeric form.

Mouse L fibroblasts and other mammalian cells are killed by the translation inhibitor hygromycin B. We have modified the gene conferring resistance against hygromycin B in E. coli in such a way that it can be transcribed in mammalian cells from the promoter of the HSVtk gene. The resulting plasmid, pHMR272, was transfected into mouse L fibroblasts and HeLa cells by the calcium phosphate method and upon selection produced clones resistant against hygromycin B. The transfection rate was similar to that obtained with other selective markers. This plasmid is a useful addition to the relatively small number of dominant selectable markers available for mammalian cells.

A series of ColE1 and pSC101 cosmid vectors have been constructed suitable for cloning large stretches of DNA. All contain a single BamHI site allowing cloning of Sau3A, MboI, BglII, BclI , and BamHI-generated fragments. These vectors have the following characteristics: (i) they are relatively small (1.7-3.4 kb); (ii) the BamHI cloning site is flanked by restriction enzyme sites enabling direct cloning of unfractionated insert DNA without generating multiple insert or vector ligation products [ Ish - Horowitz and Burke, Nucl . Acids Res. 9 (1981) 2989-2998]; (iii) two vectors ( pHSG272 and pHSG274 ) contain a hybrid Tn5 KmR/ G418R gene which is selectable in both prokaryotic and eukaryotic cells, making them suitable for transferring DNA into eukaryotic cells, and (iv) the different prokaryotic selectable markers available in the other vectors described facilitate cosmid rescue of the transferred DNA sequences from the eukaryotic cell: CmR, ApR, KmR, ( pHSG429 ), CmR, ( pHSG439 ), colicin E1 immunity ( pHSG250 ), (v) the cosmid pHSG272 was used successfully to construct a shuttle vector based on the BPVI replicon [ Matthias et al., EMBO J. 2 (1983) 1487-1492].

We describe the construction of a bovine papilloma virus‐based vector (pCGBPV9) which contains a dominant selectable marker and replicates autonomously in both mouse and Escherichia coli cells. This vector contains the complete bovine papilloma virus genome, a ColE1 replication origin and a dominant selectable marker conferring resistance to kanamycin in bacteria and G418 in eukaryotic cells. A high number of G418R colonies are obtained after transfer of pCGBPV9 into mouse C127 cells. These G418R colonies contain vector DNA which replicates autonomously at approximately 10‐30 copies per cell. The molecules are in most cases unrearranged and can be rescued into E. coli cells by bacterial transformation.

Mouse cells deficient in the enzyme hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) have been transfected with total human DNA, and cells producing human enzyme were isolated by growth in selective medium. DNA from several such cell lines has been used to generate secondary transfectants that make human HPRT. Blots of the DNA of these secondary cells have been hybridized with total human DNA probes or with cloned human Alu sequences, and one of several common bands has been cloned in pBR322. Colonies of transformed Escherichia coli containing human sequences were detected by their homology with human DNA, and subclones of resulting recombinant plasmids were prepared. Two subclones free of Alu sequences were found to contain human sequences that hybridized to human X chromosome DNA. One of these, pBR1.5, also hybridized to a single RNA band on gel blots of human and secondary transfectant cytoplasmic poly(A)+RNA but not to RNA from the parent mouse cell line. These results indicate that these clones represent human HPRT gene fragments. This has been confirmed by using pBR1.5 as a probe to isolate an authentic and expressible human HPRT cDNA clone from a library prepared by H. Okayama and P. Berg.

This chapter describes the use of the λ phage promoter PL to promote gene expression in hybrid-plasmid-cloning vehicles. The expression of prokaryotic structural genes in bacteria is dependent on the deoxyribonucleic acid (DNA) sequences upstream from these genes that do not code for a polypeptide chain, for example, promoters, operators, and ribosome-binding sites. Physical separation in vitro of a particular structural gene and its regulatory sequences may be possible by the cleavage of the DNA with a suitable restriction endonuclease at a restriction site between these two DNA sequences. This chapter describes the construction and use in Escherichia coli (E. coli) of plasmid cloning vehicles designed to promote the expression of gene inserts. DNA fragments that contain genes deleted for their own promoters can be inserted into any one of the four different single restriction sites on these vehicles. The transcription of these genes occurs from the strong phage λ promoter PL located on the vehicles, from which ribonucleic acid (RNA) polymerase can read into the inserted fragments. These vehicles should be useful in promoting high levels of expression of prokaryotic genes that are weakly expressed in their natural genetic environment or in promoting the transcription of eukaryotic genes that are poorly expressed in E. coli.